Mechanisms for polycyclic aromatic hydrocarbon degradation by ligninolytic fungi.

Ligninolytic fungi accomplish the partial degradation of numerous aromatic organopollutants. Their ability to degrade polycyclic aromatic hydrocarbons (PAHs) is particularly interesting because eukaryotes were previously considered to be unable to cleave fused-ring aromatics. Recent results indicate that extracellular peroxidases of these fungi are responsible for the initial oxidation of PAHs. Fungal lignin peroxidases oxidize certain PAHs directly, whereas fungal manganese peroxidases co-oxidize them indirectly during enzyme-mediated lipid peroxidation.

The ligninolytic fungi that cause white-rot of wood degrade a wide variety of organopollutants. The compounds metabolized include chlorophenols, chloroanilines, pesticides such as DDT and methoxychlor, and polycyclic aromatic hydrocarbons (1,2). To varying degrees, pollutants in these groups are co-oxidized by the fungi to give CO2 and largely uncharacterized polar metabolites. The xenobiotic oxidations of white-rot fungi are not rapid or efficient, but they are very nonspecific. Moreover, many white-rot fungi are natural inhabitants of soil litter. These considerations make ligninolytic fungi attractive candidates for use in low tech bioremediation programs (3). Little is known about the mechanisms that whiterot fungi employ for organopollutant oxidation.
Xenobiotic metabolism in white-rot fungi usually parallels ligninolytic metabolism. Most recent work on ligninolysis has been done with a thermotolerant whiterotter, Phanerochaete chrysosporium, and we now have a fairly extensive understanding of this basidiomycete's ligninolytic mechanisms (4,5). Ligninolysis is oxidative, is stimulated by high oxygen levels in the culture medium, and is part of the organism's secondary metabolism; it occurs only when some nutrient, usually nitrogen, is limiting. This paucity of nutrients is the normal situation in rotting wood as well as in many soils. Lignin degradation is necessarily extracellular because lignin is a macromolecular, amorphous, highly branched, waterinsoluble polymer of phenylpropane subunits. The major initial ligninolytic reaction in P. chrysosporium is oxidative cleavage of the polymer's propyl sidechain between Co and Cp ( Figure 1).
The initial depolymerization of lignin by P. chrysosporium is thought to be catalyzed by a group of related extracellular enzymes that are secreted by the fungus under ligninolytic culture conditions. These enzymes are unusually oxidizing, ferric hemoprotein peroxidases that differ significantly from commonly studied peroxidases, such as the enzyme from horseradish, in that they oxidize a much broader range of aromatic substrates. For example, these lignin peroxidases (LiPs) cleave nonphenolic lignin model dimers between Ca and C of their propyl sidechains to yield benzylic alcohols and aldehydes as end products, they oxidize polymethoxylated benzyl alcohols to benzaldehydes, and they oxidize various alkoxybenzenes to give benzoquinones and alkanols. A decade of has demonstrated a unified mechanism for these diverse enzymatic oxidations of aromatic compounds; they occur in one-electron steps, and postenzymatic reactions of the resulting aryl cation radicals account for the end products that are observed.
LiPs also catalyze the oxidation of certain aromatic pollutants, including various substituted phenols and polycyclic aromatic hydrocarbons (PAHs) (2), and could accordingly play a role in the xenobiotic metabolism of white-rot fungi. For example, certain PAHs are oxidized when they are incubated with LiP and H202 (6,7), with the susceptibility of a given compound to enzymatic attack depending in a straightforward way on its ionization potential (7). PAHs such as benz[a]anthracene, pyrene, and anthracene, which have ionization potentials < 7.35 eV, are LiP substrates, whereas PAHs such as phenanthrene and benzo[e]pyrene, which have ionization potentials >7.35 eV, are not ( Table 1). The products of these enzymatic PAH oxidations are PAH quinones. For example, benzo[a]pyrene is oxidized to its 1,6-, 3,6-, and 6,12-quinones, pyrene to its  experiments have shown, in the case of pyrene oxidation, that the new oxygens in the product quinones derive from water, presumably via nucleophilic attack of the solvent on a cationic reaction intermediate (7).
The PAHs present a particularly interesting research problem because their biological ring cleavage was previously considered a purely bacterial phenomenon, and ligninolytic fungi are the only eukaryotes so far known to degrade them. Anthracene will serve as an example: the initial steps of its bacterial metabolism (in pseudomonads) involve a dioxygenase-catalyzed oxidation to anthracene cis-1,2-dihydrodiol, further oxidation to 1,2-dihydroxyanthracene, and subsequent extradiol (meta) ring-fission (8). Previously studied fungi (e.g., Cunninghamella elegans), by contrast, use monooxygenases to activate the aromatic ring. The initial product in this case is anthracene-1,2-oxide, which can isomerize nonenzymatically to yield 1or 2-anthrol or undergo enzymatic hydration to give anthracene trans-1,2-dihydrodiol but is not a precursor for ring cleavage (8,9). Neither of these pathways is likely to occur as a function of fungal ligninolytic metabolism, which ought to lead to the degradation of anthracene via 9,10anthraquinone.
To determine whether such a new pathway exists, we investigated anthracene metabolism in P. chrysosporium (10). 14Clabeled anthracene and anthraquinone (0.2 pM) were supplied to ligninolytic (nitrogen-limited) cultures, and it was shown that '4Co2 evolution from the two compounds was similar (-13% in 14 days). The major neutral product of anthracene oxidation in vivo was anthraquinone, as shown by high pressure liquid chromatography (HPLC) and thin-layer chromatography (TLC) analysis of extracted cultures and also by isotope dilution experiments. The abiotic oxidation of anthracene to the quinone was, by contrast, negligible over the time of the experiments. Both anthracene and anthraquinone were cleaved in culture to the same ring-fission product, phthalic acid, as demonstrated again by HPLC and TLC. Isotope dilution experiments confirmed this identification, showed that anthracene and anthraquinone (2.0 pM initial concentration) gave phthalate in similar yield (12-13% in 7 days), and showed that anthraquinone cleavage occurred only in ligninolytic cultures. These results show that the major pathway for anthracene degradation in P. chrysosporium proceeds via the quinone to phthalic acid ( Figure 2) and strongly support the hypothesis that LiPs catalyze the first step of anthracene degradation in this ligninolytic fungus.
Some aromatic pollutants that are resistant to attack by LiPs are, nevertheless, degraded by P. chrysosporium. Certain PAHs fall within this group, phenanthrene being a notable example (11). Since the mechanisms responsible for these oxidations were completely unknown, we undertook the first detailed study of phenanthrene metabolism in Phanerochaete (12). The fungus oxidized [14C]phenanthrene (2.0 pM initial concentration) at its 9-and 10positions to give 10 to 15% yields of a ring-fission product, 2,2'-diphenic acid, which was identified in chromatographic and isotope dilution experiments ( Figure  3). Diphenic acid formation from phenanthrene was somewhat greater in low-nitro-  gen (ligninolytic) cultures than in highnitrogen (nonligninolytic) cultures, and did not occur in uninoculated cultures.
The presence of diphenic acid as a major oxidation product suggested that phenanthrene might be oxidized in vivo via the 9,1 0-quinone. Phenanthrenequinone could not be detected in cultures that were metabolizing phenanthrene but was cleaved to diphenic acid more rapidly than the PAH was in culture; therefore phenanthrenequinone is a plausible intermediate.
Subsequent work has shown that phenanthrene oxidation to diphenic acid by P. chrysosporium is a consequence of peroxidase-mediated lipid peroxidation. Most ligninolytic fungi secrete peroxidases whose roles are to oxidize Mn2+ to Mn , which acts as a low molecular weight diffusible ligninolytic oxidant at locations remote from the enzyme active site (13). These manganese peroxidases (MnPs) also support Mn-dependent lipid peroxidation, and in vitro, MnP oxidizes phenanthrene to diphenic acid in a slow reaction that requires Mn2+, oxygen, and unsaturated fatty acids (14). It is still not clear whether the proximal oxidant of phenanthrene is the peroxidase, a lipoxyradical, or a higher valent Mn species. Current efforts in our laboratory are therefore aimed at elucidating the reaction mechanism, which may be of general importance in the xenobiotic metabolism of white-rot fungi.